Effects Of Maternal Obesity On Hepatic Lipid Metabolism In Offspring And The Regulation Of CircRNA＿0000660

Background:Maternal obesity could cause dyslipidemia in the liver of the offspring in early life,thereby increasing the risk of the offspring suffering from related chronic diseases.The research mechanism of maternal obesity-induced liver lipid metabolism disorder in the offspring mainly focused on the inflammatory response or oxidative stress,and the mechanism of epigenetic regulation of non-coding RNA(ncRNA)was less studied.Circular RNA(circRNA)is a special endogenous ncRNA which plays an important role in the regulation of gene expression.It is unclear whether circRNA is involved in maternal obesity leading to "metabolic recombination" of the offspring’s liver.Autophagy is an important metabolic pathway.It participates in the degradation of lipids in liver and maintains the balance of lipid metabolism.However,the regulatory effect of autophagy on lipid metabolism and its molecular mechanism are still insufficient in this model.Purpose:This study aimed to explore the following issues:1.The effect of maternal obesity on lipid metabolism in the liver of the offspring;2.Whether maternal obesity affects liver lipid metabolism by changing the expression of circRNA in the liver of the offspring;3.As a key differentially expressed gene,circRNA＿0000660 regulates the lipid metabolism of the progeny liver through which regulatory mechanism?Methods:1.Establishment of animal model and collection of tissue:C57/BL6 female mice were randomly divided into two groups and fed on high-fat diet or normal diet for 12 weeks.The high-fat induced obesity model was established successfully according to body weight and serum lipids index.Then the mice were mated with male healthy mice and delivered freely.The offspring mice weaned at the third week.The weight of the mice was monitored every week.When the mice were weaned,the blood was collected from orbital venous plexus.The liver and adipose tissue were collected.2.Detection of lipid metabolism indicators including total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL)content in offspring;H&E staining was used to observe the liver morphology of offspring;oil red O staining was used to observe the lipid deposition in offspring.3.CircRNA expression analysis:using high-throughput sequencing(RNA-Seq)to identify the expression of circRNA in liver tissues of the two groups;Analyze the circRNA expression profile and screen out differently expressed circRNA;GO and KEGG analysis were perform to identified the functional clustering on the host genes;using bio informatics methods to predict the downstream target genes of differential circRNA and construct a circRNA-miRNA-mRNA regulatory network;identified the hub circRNA and predict its potential regulatory pathways through analyzing mRNA and circRNA.4.Verification of circRN A-miRNA-mRNA regulatory pathway:using real-time PCR(qRT-PCR),Western Blot(WB)to detect circRNA and its downstream target genes in the liver tissues of offspring;using palmitic acid(PA)to induce lipid deposition in AML12 cells,then using siRNA and miRNA inhibitor to interfere with the expression of circRNA and miRNA to verify the regulation of circRNA on downstream targets;dual luciferase report experiment was used to verify the binding sites between circRNA,miRNA and mRNA.5.CircRNA regulate autophagy:Using qRT-PCR,WB,immunofluorescence to detected the expression of LC3,p62 and p-mTOR which were all indicator for autophagy in liver tissues and AML12 cells;Knockdown circRNA＿0000660 with siRNA and detected the expression levels of LC3,p62 and mTOR;using mTOR signaling pathway inhibitor rapamycin(Rapa)and siRNA in AML12 cells and detected lipid deposition status.Results:1.Maternal obesity leads to lipid deposition in liver of offspring:the weight of offspring from obesity maternal is higher than the control group from 1st to 3th week;the level of TC,TG and LDL are higher than control group at the third week,;H&E staining results showed that the liver morphology of the offspring of obesity mice had obvious vacuole-like structures;oil red O results showed that the liver of the treated group had obvious lipid deposition.2.Maternal obesity changes the circRNA expression profile of the liver of the offspring:3651 circRNAs were identified in the liver of the two groups of mice,including 231 differential circRN As,121 up-regulated,and 110 down-regulated from RNA-Seq;KEGG pathway enrichment analysis showed that the host gene of circRNA mainly enriched in the relevant pathways of lipid metabolism;circRNA＿0000660 was identified as the hub gene,and may regulate lipid metabolism in the offspring of mice through circRN A＿0000660miRNA＿693＿Igfbp1 regulation mechanism.3.Verification of circRNA＿0000660 regulatory mechanism:qRT-PCR results showed that the expression of circRNA＿0000660 and Igfbpl decreased in treated group,which was consistent with the results of RNA-Seq.The expression of circRNA＿0000660 and Igfbpl were decreased in PA induced AML12 cell compared with the control group;the expression of Igfbpl was further significantly reduced after treated with siRNA,and the results of oil Red O staining showed that the lipid deposition increased;dual luciferase reporter showed that circRNA＿0000660 could bind with miR＿693 and miR＿693 could bind with Igfbp1.4.The regulatory effect of circRNA＿0000660 on autophagy:qRT-PCR,WB and immunofluorescence results showed that the expression level of liver autophagyrelated indicators LC3II in the treated group was significantly decreased,and the expression level of p62 was significantly increased.In the PA induced AML12,the level of autophagy decreased;after using siRNA in AML12 cells,the level of autophagy further decreased,suggesting that circRNA＿0000660 could regulate autophagy;qRT-PCR and WB results showed that expression of p-mTOR was increased in treated mice and AML12 cell;expression of p-mTOR further increased after treated with siRNA,Rapa could induce autophagy and decrease lipid deposition,but lipid deposition increased when siRNA treated.Conclusion:Maternal obesity could induce disorder of hepatic lipid metabolism and higher weight in the offspring.Maternal obesity inhibits the expression of circRNA＿0000660 in liver of offspring.CircRNA＿0000660 could inhibit the expression of Igfbpl by binding with miRNA＿693.Furthermore,circRNA＿0000660 could regulate the level of autophagy in liver through the mTOR signaling pathway and result in lipid metabolism disorder in liver of offspring.